The present invention relates generally to proteins useful for the treatment of gram-negative bacterial infections and specifically to the neutralization of the effects of lipopolysaccharide (LPS) which is also known as endotoxin. LPS is a major component of the outer membrane of gram-negative bacteria and consists of serotype-specific O-side chain polysaccharides linked to a conserved region of core oligosaccharide and lipid A. LPS is an important mediator in the pathogenesis of septic shock and is one of the major causes of death in intensive-care units in the United States. It has been observed that exposure to LPS during sepsis stimulates an immune response in monocytes and macrophages that results in a toxic cascade resulting in the production of tumor necrosis factor (TNF) and other proinflammatory cytokines. Morrison and Ulevitch, Am. J. Pathol., 93:527 (1978). Endothelial damage in sepsis probably results from persistent and repetitive inflammatory insults. Bone, Annals Int. Med. 115:457 (1991).
LPS-binding proteins have been identified in various mammalian tissues. Among the most extensively studied of the LPS-binding proteins is bactericidal/permeability-increasing protein (BPI), a basic protein found in the azurophilic granules of polymorphonuclear leukocytes. Human BPI protein has been isolated from polymorphonuclear neutrophils (PMNs) by acid extraction combined with either ion exchange chromatography or E. coli affinity chromatography. Weiss et al., J. Biol. Chem., 253:2664 (1978); Mannion et al., J. Immunol. 142:2807 (1989).
The holo-BPI protein isolated from human PMNs has potent bactericidal activity against a broad spectrum of gram-negative bacteria. This antibacterial activity appears to be associated with the amino terminal region (i.e. amino acid residues 1-199) of the isolated human holo-BPI protein. In contrast, the C-terminal region (i.e. amino acid residues 200-456) of the isolated holo-BPI protein displays only slightly detectable anti-bacterial activity. Ooi et al., J. Exp. Med., 174:649 (1991). Human DNA encoding BPI has been cloned and the amino acid sequence of the encoded protein has been elucidated. Gray et al., J. Biol. Chem., 264:9505 (1989). Amino-terminal fragments of BPI include a natural 25 Kd fragment and a recombinant 23 Kd, 199 amino acid residue amino-terminal fragment of the human BPI holoprotein referred to as rBPI23. See, Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992). In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI23) having the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in SEQ ID NOS: 11 and 12 taken from Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein referred to herein as rBPI has also been produced having the sequence set out in SEQ ID NOS: 11 and 12 taken from Gray et al., supra, with the exceptions noted for rBPI23. See also, Elsbach et al., U.S. Pat. No. 5,198,541 the disclosure of which is hereby incorporated by reference. In addition to its bactericidal effects, BPI has been shown to neutralize the toxic and cytokine-inducing effects of LPS to which it binds.
Lipopolysaccharide binding protein (LBP) is a 60 kD glycoprotein synthesized in the liver which shows significant structural homology with BPI. Schumann et al. disclose the amino acid sequences and encoding cDNA of both human and rabbit LBP. Like BPI, LBP has a binding site for lipid A and binds to the LPS from rough (R-) and smooth (S-) form bacteria. Unlike BPI, LBP does not possess significant bactericidal activity, and it enhances (rather than inhibits) LPS-induced TNF production. Schumann et al., Science, 249:1429 (1990). Thus, in contrast to BPI, LBP has been recognized as an immunostimulatory molecule. See, e.g., Seilhamer, PCT International Application WO 93/06228 which discloses a variant form of LBP which it terms LBP-xcex2.
One of the normal host effector mechanisms for clearance of bacteria involves the binding to and subsequent phagocytosis by neutrophils and monocytes. As part of this process, bacteria are exposed to bactericidal and bacteriostatic factors, including oxygen radicals, lysosomal enzymes, lactoferrin and various cationic proteins. LBP opsonizes LPS-bearing particles and intact Gram-negative bacteria, mediating attachment of these LBP-coated particles to macrophages. Wright et al., J. Exp. Med. 170:1231 (1989). The attachment appears to be through the CD14 receptor of monocytes which binds complexes of LPS and LBP. Wright et al., Science 249:1431 (1990). Anti-CD14 mAbs have been shown to block the synthesis of TNF by whole blood incubated with LPS. Wright et al. Science 249:1431 (1990). Interaction of CD14, which is present on the surface of polymorphonuclear leukocytes as well as monocytes, with LPS in the presence of LBP has been shown to increase the adhesive activity of neutrophils. Wright et al., J. Exp. Med. 173:1281 (1991), Worthen et al., J. Clin. Invest. 90:2526 (1992). Thus, while BPI has been shown to be cytotoxic to bacteria and to inhibit proflammatory cytokine production stimulated by bacteria, LBP promotes bacterial binding to and activation of monocytes through a CD14-dependent mechanism.
LPS, either directly or by inducing proinflammatory cytokines such as IL-1 and TNF, induces the expresion of adhesion molecules including CD54 (intercellular adhesion molecule-1, ICAM-1) and E-selectin (endothelial-leukocyte adhesion molecule-1, ELAM-1) on endothelial cells, and thereby increases binding of leukocytes in vitro. Schleimer and Rutledge, J. Immunol. 136:649 (1986); Pohlman et al., J. Immunol. 136:4548 (1986); Bevilacqua et al., J. Clin. Invest. 76:2003 (1985); Gamble et al., Proc. Natl. Acad. Sci. USA. 82:8667 (1985); Smith et al., J. Clin. Invest. 82:1746 (1988); and Bevilacqua et al., Proc. Natl. Sci. USA 84:9238 (1987). However, as CD14 has not been detected on the surface of endothelial cells (Beekhuizen et al., J. Immunol. 147:3761 (1990)) and no other receptor for LPS on endothelial cells has been identified, a different mechanism may exist whereby LPS can affect the endothelium.
Soluble CD14, found in serum (Bazil et al., Eur. J. Immunol. 16:1583 (1986)), has been hypothesized to be responsible for transmitting the LPS signal to endothelial cells. Specifically, soluble CD14 has been shown to mediate a number of LPS-dependent effects on endothelial cells, including E-selectin and VCAM expression, IL-1, IL-6 and IL-8 secretion, and cell death. Frey et al., J. Exp. Med. 176:1665 (1992): Pugin et al., Proc. Natl. Acad. Sci. USA. 90:2744 (1993).
Recent studies have shown that soluble CD14 is involved in the LPS-mediated adhesion of neutrophils to endothelial cells. Anti-CD14 mAbs were able to completely inhibit the adhesion induced by LPS, indicating that the contribution of other CD14-independent LPS receptors to these effects is minimal. The protein(s) on the endothelial cells that soluble CD14 might associate with to transduce the LPS signal remains to be identified. LBP has been shown to be involved in the signal transduction of LPS through soluble CD14; however, at high concentrations of LPS or soluble CD14, LBP does not further enhance the response of endothelial cells to LPS (Pugin et al., Proc. Natl. Acad. Sci. USA. 90:2744 (1993).
Larrick et al., Biochem. and Biophysical Res. Commun., 179:170 (1991) relates to a cationic protein obtained from rabbit granulocytes which is identified as CAP18. CAP18 is identified as bearing no sequence homology with either BPI or LBP. In the course of their disclosure, Larrick et al. characterize other publications which discuss the structure of proteins including LBP and incorrectly attribute to the Wright et al., supra disclosure the speculation that xe2x80x9cLBP is believed to be composed of two regions: an amino-terminal domain that binds to LPS and a carboxy-terminal domain that may (emphasis supplied) mediate binding of the LBP-LPS complex to the CD14 receptor on leukocytes.xe2x80x9d
Ulevitch, PCT International Application WO 91/01639 discloses methods and compositions for treatment of sepsis comprising administering anti-CD14 antibodies. The published application also describes xe2x80x9cLBP peptide analogsxe2x80x9d at page 17 which are stated to be polypeptides capable of competitively inhibiting the binding of LPS-LBP complexes to CD14 expressed on the surface of monocyte derived macrophages. The sequences of the three disclosed xe2x80x9cLBP peptide analogsxe2x80x9d show 90 to 100% homology with CD14 polypeptide sequences and no homology with LBP sequences.
Ulevitch et al., U.S. Pat. No. 5,245,013 discloses a lipopolysaccharide binding protein which binds to Gram-negative bacterially secreted LPS and retards in vitro binding of LPS to high density lipoprotein.
Marra, PCT International Application WO 92/03535 discloses various chimeric BPI molecules including an rLBP/BPI chimeric molecule designated LBP25K/BPI30K [LBP(1-197)/BPI(200-456)] and comprising the first 197 amino acid residues of LBP and amino acid residues 200-456 of BPI wherein the coding sequence for the amino-terminal 25 kD portion of LBP was linked to the coding sequence for the carboxy-terminal portion of the BPI protein by virtue of an engineered ClaI site within the coding sequence. The resulting molecule reacted positively in an ELISA assay utilizing anti-BPI protein antibodies and also reacted positively in an endotoxin binding assay. Rogy et al., J. Clin. Immunol., 14: 120-133 (1994) describes experiments utilizing the LBP(1-197)/BPI(200-456) molecule wherein animals treated with the molecule in a primate bacteremia model demonstrated decreased LPS levels compared to controls, but still developed the sequalae of septic shock. For example, no significant reduction in endotoxin mediated cytokine synthesis was observed in endotoxin-treated baboons to whom the compound was administered.
There exists a need in the art for LPS binding and neutralizing proteins which lack CD14-mediated immunostimulatory properties, including the ability to mediate LPS activity through the CD14 receptor.
The present invention provides novel biologically active polypeptide derivatives of Lipopolysaccharide Binding Protein (LBP), including LBP derivative hybrid proteins, which are characterized by the ability to bind to LPS and which lack CD14-mediated immunostimulatory properties, including the ability of LBP holoprotein to mediate LPS activity via the CD14 receptor. More particularly, LBP protein derivatives including LBP derivative hybrid proteins according to the invention lack those carboxy terminal-associated elements characteristic of the LBP holoprotein which enable LBP to bind to and interact with the CD14 receptor on monocytes and macrophages so as to provide an immunostimulatory signal to monocytes and macrophages.
Presently preferred LBP protein derivatives are characterized by a molecular weight less than or equal to about 25 kD. Particularly preferred LBP protein derivatives of the invention are LBP fragments comprising an amino-terminal region of LBP (e.g., amino acid residues 1-197). A molecule comprising the first 197 amino terminal residues of LBP and designated rLBP25 exemplifies the derivatives of the invention. This particular derivative includes amino acid regions comprising LBP residues 17 through 45, 65 through 99 and 141 through 167 which correspond to respective LPS binding domains (e.g., residues 17 through 45, 65 through 99 and 142 through 169) of Bactericidal/Permeability-Increasing protein (BPI).
LBP derivative hybrid proteins of the invention comprise hybrids of LBP protein sequences with the amino acid sequences of other polypeptides and are also characterized by the ability to bind to LPS and the absence of CD14-mediated immunostimulatory properties. Such hybrid proteins can comprise fusions of LBP amino-terminal fragments with polypeptide sequences of other proteins such as BPI, immunoglobulins and the like. Preferred LBP/BPI hybrids of the invention comprise at least a portion (i.e., at least five consecutive amino acids and preferably ten or more amino acids) of an LPS binding domain of BPI. One preferred LBP derivative hybrid protein of the LBP/BPI type comprises an amino-terminal LBP amino acid sequence selected from within the amino terminal half of LBP (e.g. within amino acid residues 1-197 of LBP) in which one or more portions of that sequence is replaced by the corresponding sequence of BPI selected from within the amino terminal half of BPI (e.g., within amino acid residues 1-199 of BPI). Another preferred LBP derivative hybrid protein comprises a fusion of amino terminal portions of LBP and heavy chain regions of IgG. Other LBP derivative hybrid proteins comprise LBP amino acid sequences into which all or portions of LPS binding domains of e.g., BPI or other LPS binding protein have been inserted or substituted for all or part of an LPS binding region of LBP. Preferred LBP derivative hybrid proteins include those in which all or portions of the previously-noted amino terminal LPS binding domains of BPI replace the corresponding region within LBP.
LBP protein derivatives and LBP derivative hybrid proteins of the invention are expected to display one or more advantageous properties in terms of pharmacokinetics, LPS binding, LPS neutralization and the like.
The present invention further provides novel pharmaceutical compositions comprising the LBP protein derivatives and LBP derivative hybrid proteins along with pharmaceutically acceptable diluents, adjuvants, and carriers and correpsondingly addresses the use of LBP protein derivatives and LBP derivative hybrid proteins in the manufacture of medicaments for treating gram negative bacterial infections and the sequelae thereof.
Polypeptides of the invention may be synthesized by assembly of amino acids. In addition, the invention provides DNA sequences, plasmid vectors, and transformed cells for producing the LBP protein derivatives and LBP hybrid derivative proteins of the invention.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.